Propagation of allosteric changes through the catalytic-regulatory interface of Escherichia coli aspartate transcarbamylase

Each of two previously isolated strains of Escherichia coli containing a single nonsense codon within the pyrB gene was suppressed with four different nonsense suppressors. The kinetic analysis using crude extracts of these nonsense-suppressed strains indicated that the mutant aspartate transcarbamylases had altered cooperativity and affinity for aspartate as judged by the substrate concentration at half of the maximal velocity. Both pyrB genes were cloned and then sequenced. In both cases, a single base change was identified which converted a glutamine GAC codon into a TAC nonsense codon. Both mutations occurred in the catalytic chain of aspartate transcarbamylase and were identified at positions 108 and 246. The glutamine at position 108 in the wild-type structure is located at the interface between the catalytic and regulatory chains and is involved in a number of interactions with backbone and side chains of the regulatory chain. The glutamine at position 246 in the wild-type structure is located in the 240s loop of the enzyme. Two additional mutant versions of aspartate transcarbamylase were created by site-directed mutagenesis to further investigate the 108-position in the structure, a glutamine to tyrosine substitution at position 108 of the catalytic chain, and an asparagine to glycine change at position 113 of the regulatory chain, a residue which interacts directly with glutamine-108 in the wild-type structure. Both mutant enzymes have reduced affinity for aspartate. However, the Tyr-108 mutant enzyme exhibits a reduced Hill coefficient while the Gly-113 enzyme exhibits an increased Hill coefficient. The response to the allosteric effectors ATP and CTP is also changed for both the mutant enzymes.(ABSTRACT TRUNCATED AT 250 WORDS)